Summary/Abstract Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are essential for rhythmic activity in the heart and brain. Mutations in HCN channels are linked to heart arrhythmia and epilepsy. HCN channels belong to the family of voltage-gated K+ (Kv) channels. Hyperpolarization-activated HCN channels and depolarization- activated Kv10.1 (EAG) channels have very similar tetrameric structures with six transmembrane segments (S1-S6) per subunit: S1-S4 form the voltage-sensing domain (VSD) and S5-S6 form the pore domain (PD). In both Kv and HCN channels, S4 is the positively charged voltage sensor and the C-terminal part of S6 forms the gate. However, why Kv channels are activated by depolarization whereas HCN channels are activated by hyperpolarization is not clear. Using voltage clamp fluorometry, FRET, and cysteine accessibility and crosslinking, we will here measure the movement of S4 and the gate in HCN channels to determine the mechanism of activation of HCN channels by hyperpolarization. Our main hypothesis is that small differences in free energy between the closed and open states, due to different interactions between S4 and the pore in the different channels determines whether an ion channel opens by hyperpolarizations or depolarizations. Using recent structures of HCN and HCN-related channels as guide, we will mutate residues at the voltage sensor-pore domain interface and measure the effect of these mutations on voltage activation in HCN channels. In support of our main hypothesis, we show that mutations of only two residues located at the interface between this region of S4 and the PD reverse the voltage dependence of HCN channels so that the channels now open upon depolarization instead of hyperpolarization. We also hypothesize that the main S4 movement is not sufficient to open the gate, but that a second S4 movement is necessary for gate opening. We will measure these two different voltage sensor movements in HCN channels using membrane- impermeable cysteine reagents or fluorophores attached to S4, to determine what conformational change of S4 is necessary to open the gate in HCN channels. Using the recent structures of HCN and HCN-related channels, we will created molecular models of the different states of HCN channels to suggest a specific conformational change of S5 and S6 during gate opening. We will test the hypothesized gate movement using cysteine crosslinking between different channel domains. We will also measure conformational changes using unnatural fluorescent ANAP as the donor and transition metals as acceptors to conduct transition metal FRET between different channel domains. A better understanding of HCN channel gating will aid in development of better anti-arrhythmic and anti-epileptic drugs targeting HCN channels.